Abstract

Background: Heritable functional genetic variation in the VEGF pathway may impact on tumor angiogenesis and, hence, clinical cancer outcomes. However, there is very limited data currently available on which common germline variants affect gene expression in the tumor vasculature. Thus, the purpose of this study is to fill this critical gap in the knowledge. To achieve this aim, we used tumor samples to characterize the mRNA expression of several key genes in the VEGF pathway and then examined genetic associations with candidate SNPs. We have used several different approaches to generate a list of candidate SNPs for analysis, including: the use of bioinformatic tools, our previous study of gene resequencing of VEGF-pathway genes and genetic associations with mRNA levels in lymphoblastoid cell lines (LCLs) (Paré-Brunet, AACR 2008;#4332), and the identification of functional variants from the literature.

Methods: The mRNA expression of HIF1A, KRAS (isoforms A and B), PGF, VEGFA (total and plus the 165 isoform) and VEGFB was measured by quantitative PCR in 91 European NSCLC specimens and mRNA levels were normalized using the expression of 18S. Common putatively regulatory or functional SNPs in these genes were identified using our previous study, the FastSNP bioinformatic web tool (http://fastsnp.ibms.sinica.edu.tw), and literature searches. Candidate SNPs were genotyped in the NSCLC cohort using TaqMan assays and genotype-expression associations were analyzed by linear regression, correcting for clinical characteristics of patients.

Results : We quantified NSCLC tumoral mRNA expression and found PGF mRNA levels were significantly greater (p<0.001) than those of any other gene and were followed in decreasing order of expression by VEGFB, KRAS (A and B isoforms), VEGFA (total and 165 isoform) and HIF1A. We genotyped 14 SNPs using matching DNA samples and three SNPs significantly associated with mRNA levels in NSCLC tumors: rs10842513 (minor allele frequency (MAF)=0.10), rs3025006 (MAF=0.32) and rs1573060 (MAF=0.38). rs10842513 correlated with higher KRAS (isoform B) mRNA levels after controlling for tumor histology (p=0.007), in agreement with the association between this variant and KRAS levels in LCLs; rs3025006 associated with lower total VEGFA expression (p=0.020); and rs1573060 correlated with both lower total VEGFA levels (p=0.021), as well as with lower expression of the 165 isoform (p=0.024).

Conclusion: We have successfully found common genetic variants which associate with mRNA expression from tumor samples. This study provides crucial knowledge as functional genetic variants may affect clinical cancer outcomes. Indeed, one of SNPs we identified, rs10842513, is known to associate with poorer survival in ovarian cancer (Quaye et al., Clin Cancer Res 2008;14: 5833). Such clinical associations will be examined in future studies.